EP4326735A1 - Compositions pour la synthèse chimique de peptides - Google Patents

Compositions pour la synthèse chimique de peptides

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Publication number
EP4326735A1
EP4326735A1 EP22792712.6A EP22792712A EP4326735A1 EP 4326735 A1 EP4326735 A1 EP 4326735A1 EP 22792712 A EP22792712 A EP 22792712A EP 4326735 A1 EP4326735 A1 EP 4326735A1
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EP
European Patent Office
Prior art keywords
group
range
butyl
constituent
integer selected
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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EP22792712.6A
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German (de)
English (en)
Inventor
Cole SEIFERT
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Sederma SA
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Sederma SA
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Publication date
Application filed by Sederma SA filed Critical Sederma SA
Publication of EP4326735A1 publication Critical patent/EP4326735A1/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/30—Phosphinic acids [R2P(=O)(OH)]; Thiophosphinic acids ; [R2P(=X1)(X2H) (X1, X2 are each independently O, S or Se)]
    • C07F9/36—Amides thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/30—Phosphinic acids [R2P(=O)(OH)]; Thiophosphinic acids ; [R2P(=X1)(X2H) (X1, X2 are each independently O, S or Se)]
    • C07F9/32—Esters thereof
    • C07F9/3205—Esters thereof the acid moiety containing a substituent or a structure which is considered as characteristic
    • C07F9/3229—Esters of aromatic acids (P-C aromatic linkage)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/10—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using coupling agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0202—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-X-X-C(=0)-, X being an optionally substituted carbon atom or a heteroatom, e.g. beta-amino acids

Definitions

  • PCT/US19/33296 “Method for Solution-Phase Peptide Synthesis and Protecting Strategies Thereof,” filed on May 21 st , 2019; iv) PCT Application No. PCT/US20/15132, filed January 26 th , 2020; v) U.S. App. Ser. No. 17/104,166, filed November 25, 2020 and published as US2021/0079036 Al, entitled “Compositions and Methods for Chemical Synthesis”; and vi) U.S. Prov. Pat. App. Ser. No. 63/201,313, entitled “Compositions for Chemical Synthesis of Peptides,” filed April 23,2021; and these applications are herein incorporated by reference as examples.
  • GAP Group-Assisted Purification
  • chemistry/technology as a chemistry for organic synthesis that avoids traditional purification methods such as chromatography and/or recrystallization by purposefully introducing a well -functionalized group in the starting material or in the newly generated product.
  • GAP groups can also often be used as protecting groups to prevent undesired side-reactions during the synthesis of target molecules.
  • Such research has the potential to encompass the entire field of synthetic organic chemistry.
  • Protecting groups are found in almost every complex synthesis where multiple functional groups are present. Effective protecting groups need to be robust to a wide variety of conditions and must be added and removed with high yield. Protecting groups are used extensively in peptide synthesis, either for solid or solution phase approaches. One of the most commonly used protecting group strategies for peptide synthesis is Fmoc//Bu. Fmoc//Bu chemistry is extremely attractive for peptide synthesis for several reasons, such as the availability of starting materials, cost of production, and the comparatively mild conditions needed for deprotection. U.S. Patent No.
  • SPPS has become a standard protocol used by multiple scientific disciplines for research and manufacturing.
  • the key disadvantage of SPPS lies in the difficulty of scale-up: many polymer supports are expensive and occupy the vast majority of the mass of the material to be worked with. Coupling reactions in SPPS are also inefficient because the reactions occur on a solid-liquid interface. Additionally, after each deprotection and coupling reaction, the resin must be washed with solvent to remove any impurities generated from previous reactions, and this generates significant solvent waste that can be extremely problematic on a large scale.
  • the standard protocol for Fmoc deprotection is to stir the Fmoc-peptide in a solution of dimethylformamide (DMF) or dichloromethane (DCM) with excess piperidine, deprotecting the Fmoc group and forming NFMP in the process.
  • the ‘891 patent teaches removal of this impurity by deprotecting with 4- aminomethylpiperidine (4AMP) instead of piperidine.
  • 4AMP 4- aminomethylpiperidine
  • the 4AMP additionally scavenges the active amino acid ester and enables the removal of the 4AMP-amino- acid product from the reaction mixture via washes.
  • a significant problem with this method lies in the high cost of using 4AMP, rendering the method cost prohibitive and preventing it from being widely accepted by the industry.
  • Fmoc-based SolPPS can be seen in published patent application W02017112809A1.
  • This publication teaches the use of a C-terminus GAP protecting group, benzyl diphenylphosphine oxide (HOBnDpp), to control the solubility of the target peptide to allow for selective precipitation after each successive coupling reaction.
  • the solubility is controlled such that the growing peptide remains in an organic solvent, such as ethyl acetate or DCM, and aqueous washes are performed to remove impurities; a subsequent concentration of the organic solvent followed by mixture in an alkane solvent selectively precipitates the peptide product.
  • the present disclosure offers several advantages in the art.
  • a novel method of solution-phase peptide synthesis is presented that utilizes a modular approach to anchor synthesis that can increase solubility control over a given peptide sequence.
  • the present disclosure includes using a peptide as an anchor to synthesize a target peptide.
  • the peptide can include functionalized protecting groups that can assist in solubility.
  • the present disclosure can include a chemical composition comprising a GAP constituent, a stopper constituent, and a linker constituent.
  • the present disclosure can include a method of solution-phase peptide synthesis, and the method can include the steps of attaching a first peptide to a first amino acid; coupling one or more additional amino acids to the first amino acid to form a second peptide; and removing the first peptide from the second peptide.
  • the present disclosure can include a method of forming an anchor peptide for solution-phase peptide synthesis, and the method can include the steps of coupling a first protected amino acid to a second protected amino acid; and attaching a linker to either the first or the second protected amino acid.
  • the present disclosure can include a chemical composition, which can be selected from the group consisting of:
  • the present disclosure can include a method of peptide synthesis, and the method can include attaching a first protecting group to a first side chain of a first amino acid; coupling a second protecting group to a second side chain of a second amino acid; coupling the first amino acid with the second amino acid; attaching a linker to a terminus of the first or second amino acid; attaching the linker to a third amino acid; and coupling a fourth amino acid with the third amino acid.
  • the present disclosure can include a chemical composition comprising a GAP constituent selected from the group consisting of: (Formula A), and (Formula B); wherein: R 4 is selected from the group consisting of: H, methyl, ethyl, propyl, isopropyl, benzyl, isobutyl, sec-butyl, and tert-butyl; R 5 is selected from the group consisting of: H, methyl, ethyl, propyl, isopropyl, benzyl, isobutyl, sec-butyl, and tert-butyl; R 6 is selected from the group consisting of: -C(O)-, S, O, NH, NMe, Net, NBn, NPh, and -(CH 2 ) p -, wherein “p1” is an integer selected from the range of 0 up to and including 30; R 7 is selected from the group consisting of: - C(O)
  • R 10 is selected from the group consisting of: -C(O)-, -(CH 2 )m-, and NH, wherein “m” is an integer selected from the range of 0 up to and including 30; “j” is an integer selected from the range of 0 up to and including 30; and “k” is an integer selected from the range of 0 up to and including 30.
  • linker constituent is selected from the group consisting of:
  • Y is selected from the group consisting of: S, NH, NMe, NEt, NBn, NPh, and O;
  • V is selected from the group consisting of: S, NH, NMe, NEt, NBn, NPh, O, -C(O)-, -(CH 2 ) a1 -, wherein “al” is an integer selected from the range of 0 up to and including 30;
  • W is selected from the group consisting of: S, NH, NMe, NEt, NBn, NPh, O, -C(O)-, -(CH 2 ) a 2-, wherein “a2” is an integer selected from the range of 0 up to and including 30;
  • X is selected from the group consisting of: S, NH, NMe, NEt, NBn, NPh, O, -C(O)-, -(CH 2 ) a3 -, wherein “a3” is an integer
  • R 4 is selected from the group consisting of: H, methyl, ethyl, propyl, isopropyl, benzyl, isobutyl, sec-butyl, and tert-butyl
  • R 5 is selected from the group consisting of: H, methyl, ethyl, propyl, isopropyl, benzyl, isobutyl, sec-butyl, and tert-butyl
  • R 6 is selected from the group consisting of: -C(O)-, S, O, NH, NMe, Net, NBn, NPh, and -(CH2) q1 -, wherein “ql” is an integer selected from the range of 0 up to and including 30
  • R 7 is selected from the group consisting of: - C(O)-, S, O, NH, NMe, Net, NBn, NPh, and -(CH2) q2 -, wherein “q2” is an integer
  • R 4 is selected from the group consisting of: H, methyl, ethyl, propyl, isopropyl, benzyl, isobutyl, sec-butyl, and tert-butyl
  • R 5 is selected from the group consisting of: H, methyl, ethyl, propyl, isopropyl, benzyl, isobutyl, sec-butyl, and tert-butyl
  • R 6 is selected from the group consisting of: -C(O)-, S, O, NH, NMe, Net, NBn, NPh, and -(CH 2 ) p1 -, wherein “p1” is an integer selected from the range of 0 up to and including 30
  • R 7 is selected from the group consisting of: - C(O)-, S, O, NH
  • R 11 is selected from the group consisting of: S, O, NH, NMe, Net, NBn, and NPh
  • R 12 is selected from the group consisting of: H, methyl, ethyl, propyl, isopropyl, benzyl, isobutyl, sec- butyl, tert-butyl, and -(CH 2 ) q5 -H, wherein “q5” is an integer selected from the range of 0 up to and including 30
  • R 13 is selected from the group consisting of: H, methyl, ethyl, propyl, isopropyl, benzyl, isobutyl, sec-butyl, tert-butyl, and -(CH 2 ) q6 -H, wherein “q6” is an integer selected from the range of 0 up to and including 30
  • R 14 is selected from the group consisting of: H, methyl, ethyl, propyl
  • the present disclosure can include a chemical composition comprising a GAP constituent, a stopper constituent, and a linker constituent, wherein the GAP constituent is (Formula B), wherein: R 6 is selected from the group consisting of: -C(O)-, S, O, NH, NMe, Net, NBn, NPh, and -(CH 2 ) p1 -, wherein “p1” is an integer selected from the range of 0 up to and including 30; R 7 is selected from the group consisting of: -C(O)-, S, O, NH, NMe, Net, NBn, NPh, and -(OH) p2 -, wherein “p2” is an integer selected from the range of 0 up to and including 30; R 8 is selected from the group consisting of: -C(O)-, S, O, NH, NMe, Net, NBn, NPh, and -(CH 2 ) p3 -, wherein: R
  • the present disclosure can include a chemical composition comprising a GAP constituent, a stopper constituent, and a linker constituent, wherein the GAP constituent is
  • linker constituent is wherein Y is selected from the group consisting of: S, NH, NMe, NEt, NBn, NPh, and O; V is selected from the group consisting of: S, NH, NMe, NEt, NBn, NPh, O, -C(O)-, -(CH 2 ) a1 -, wherein “al” is an integer selected from the range of 0 up to and including 30; W is selected from the group consisting of: S, NH, NMe, NEt, NBn, NPh, O, -C(O)-, -(CH 2 ) a2 -, wherein “a2” is an integer selected from the range of 0 up to and including 30; X is selected from the group consisting of: S, NH, NMe, NEt, NBn, NPh, O, -C(O)-, -(CH 2 ) a3 -, wherein “
  • the present disclosure can include a method of solution- phase peptide synthesis, the method comprising the steps of: attaching a first peptide to a first amino acid, wherein the first peptide is an anchor peptide; coupling one or more additional amino acids to the first amino acid to form a second peptide; and removing the first peptide from the second peptide.
  • the first peptide includes a GAP constituent.
  • the first peptide is formed by coupling a first protected amino acid to a second protected amino acid and attaching a linker to either the first or the second protected amino acid, wherein the first protected amino acid is formed by attaching a first protecting group to a first side chain of a first amino acid constituent, and the second protected amino acid is formed by coupling a second protecting group to a second side chain of a second amino acid constituent. Further comprising the steps of: coupling the first amino acid constituent with the second amino acid constituent; attaching the linker to a terminus of the first or second amino acid constituent; attaching the linker to a third amino acid; and coupling a fourth amino acid with the third amino acid.
  • the present disclosure can include a method of peptide synthesis, comprising the steps of coupling an anchor to a first amino acid, and coupling a second amino acid with the first amino acid, wherein the anchor includes a compound selected from the group consisting of:
  • the anchor is coupled to a C-terminus of the first amino acid.
  • the anchor is coupled to a side chain of the first amino acid, wherein the compound is and wherein the anchor is coupled to the C-terminus of the first amino acid via a linker constituent.
  • the anchor further includes a stopper constituent. Further comprising the step of removing the anchor from the first amino acid.
  • the coupling of the second amino acid with the first amino acid occurs in 2-methyltetrahydrofuran.
  • FIG 1 A illustrates an exemplary GAP constituent in accordance with the principles of the present disclosure
  • FIG IB illustrates exemplary AA(Gap) constituents in accordance with the principles of the present disclosure
  • FIG 2 illustrates exemplary amino acid constituents in accordance with the principles of the present disclosure
  • FIG 3 illustrates exemplary linker constituents in accordance with the principles of the present disclosure
  • FIG 4 illustrates exemplary stopper constituents in accordance with the principles of the present disclosure
  • FIG. 5 illustrates an exemplary anchor molecule and/or anchor peptide with an exemplary underlying structural schematic in accordance with the principles of the present disclosure
  • FIG. 6 illustrates an exemplary anchor molecule with an exemplary underlying structural schematic in accordance with the principles of the present disclosure
  • FIG. 7 illustrates an exemplary anchor molecule and/or anchor peptide with an exemplary underlying structural schematic in accordance with the principles of the present disclosure
  • FIG 8 illustrates an exemplary modular anchor synthesis method in accordance with the principles of the present disclosure. DETAILED DESCRIPTION OF THE DISCLOSURE
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations can be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit, and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.
  • components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components.
  • the defined steps can be carried out in any order or simultaneously (except where the context excludes that possibility), and the method can include one or more other steps which are carried out before any of the defined steps, between two of the defined steps, or after all the defined steps (except where the context excludes that possibility).
  • the term “at least” followed by a number is used herein to denote the start of a range beginning with that number (which may be a range having an upper limit or no upper limit, depending on the variable being defined). For example, “at least 1” means 1 or more than 1.
  • the term “at most” followed by a number is used herein to denote the end of a range ending with that number (which may be a range having 1 or 0 as its lower limit, or a range having no lower limit, depending upon the variable being defined). For example, “at most 4” means 4 or less than 4, and “at most 40%” means 40% or less than 40%.
  • a range is given as “(an initial number) to (a subsequent number)” or “(an initial number)-(a subsequent number),” this means a range whose lower limit is the initial number and whose upper limit is the subsequent number.
  • 25 to 100 mm means a range whose lower limit is 25 mm, and whose upper limit is 100 mm.
  • amino acid is a term commonly understood in the art.
  • An amino acid in the broadest sense, can be any organic molecule containing both an amino group and a carboxylic acid group. For practical application, these molecules can generally be less than 1,000 Daltons in size, with the amino group (N-terminus) separated from the carboxylic acid group (C-terminus) by only one or two methylene units.
  • An additional feature of an amino acid can be the presence of a side chain, as a branch from the methylene unit separating the N and C termini.
  • the side chain can include practically any structural feature, and the variation in the side chain accounts for the variation in amino acids.
  • GAP amino acids can be amino acids where the side chain and/or C-terminus and/or N-terminus contains and/or is attached to one or more GAP groups.
  • GAP groups as used herein can include GAP constituents (GAP anchors) (GAP protecting groups) as further discussed below. GAP groups can include, but are not limited to, a phosphine oxide moiety.
  • the amino acid “side chain” can be a branch from the methylene unit separating the N and C termini, and can also sometimes be referred to generally as an “R” group.
  • a side chain can include practically any structural feature, and the variation in the side chain accounts for the variation in amino acids.
  • “Peptide” is a term commonly understood in the art as a biopolymer made from amino acid monomers. There must be at least two amino acids bonded together to constitute a peptide.
  • An “anchor peptide” as used herein can include a peptide that also functions as an anchor molecule - for anchor peptides, a peptide is made, which then can function as an anchor to synthesize the second, target peptide.
  • an anchor peptide can include a peptide having 20 or fewer amino acids.
  • An “anchor” as used herein can be a molecular group that is attached or designed for attachment to a target molecule (such as a peptide) for the purpose of controlling the solubility properties of that peptide during its synthesis, in order to facilitate the synthesis of the peptide in a practical fashion. Anchors can generally be removed at the end of the peptide synthesis and may not be a component of the final target molecule.
  • a “GAP anchor” as used herein can include an anchor molecule that includes a GAP group.
  • An anchor can include a GAP constituent (discussed below).
  • a GAP constituent can itself serve as an anchor.
  • the present disclosure can include structures of anchor molecule for Group-Assisted Purification Peptide Synthesis (GAP -PS).
  • GAP -PS Group-Assisted Purification Peptide Synthesis
  • an anchor molecule can be coupled to a first amino acid in a peptide sequence without causing significant racemization.
  • an anchor molecule can be stable to the conditions of amino acid coupling and Fmoc deprotection steps.
  • an anchor molecule can be cleaved from the peptide without causing racemization, either concomitantly with the side-chain protecting groups, or via an orthogonal method.
  • the anchor can provide substantial control over solubility.
  • an anchor molecule can completely exclude a protected peptide from dissolving in water or water / organic solvent mixtures where the water content is > 50%. In one embodiment, failure of an anchor molecule to do this could result in low yields due to repetitive loss of product during aqueous workup.
  • an anchor can also promote solubility of a protected peptide in the chosen organic solvent, such that the peptide can completely dissolve to form a homogenous solution at concentrations suitable for large-scale manufacture, typically on the order of 10 - 20 mL of solvent per gram of solute.
  • can anchor can be a protection group capable for “anchoring” a target molecule in a particular solvent.
  • the present disclosure can include anchors that provide significant improvement to these above-discussed properties, as well as providing additional benefits.
  • the anchors considered herein can include a GAP-protected amino acid, and in another example, the utilization of such GAP -protected amino acid to build, in a modular fashion, an anchor that can be custom-tailored to a peptide being synthesized.
  • the present disclosure can include a system that can use fast and reliable chemistry of peptide coupling and deprotection reactions to build an anchor (GAP anchor).
  • a GAP anchor can include an anchor peptide (GAP anchor peptide).
  • an anchor peptide can be an anchor having a peptide bond; in another embodiment, an anchor peptide can be any peptide; in another embodiment, an anchor peptide can be any molecule having a peptide bond that is soluble in polar organic solvents and insoluble in water. In another embodiment, an anchor peptide can be any molecule having a peptide bond that can be removably attached to a target molecule to facilitate solubility control, and subsequently removed from the target molecule. In another embodiment, anchors can be quickly synthesized that incorporate one, two, three, or more GAP amino acids for varying degrees of control over solubility that, for example, can be balanced with cost.
  • synthesis of a 30-mer peptide can be accomplished via the use of three GAP amino acids in the anchor to adequately control the solubility of a peptide this size; however, in another example, a 5-mer peptide can be achieved using an anchor with one GAP amino acid.
  • the same GAP amino acid can be synthesized as a common feedstock for both of these anchors, and can be quickly assembled as an anchor in a manner consistent with the needs of the target molecule, and in a way which balances the control required with the cost of anchor synthesis and atom economy.
  • the increased stability of the GAP amino acid anchors can allow both the coupling and deprotection reactions to be run in wet organic solvent (water content ⁇ 10%). This can, for example, eliminate the need for time-consuming, and therefore costly, drying steps which can include filtration through a solid drying agent and / or azeotropic distillation.
  • the present disclosure offers significant advantages by eliminating the need for these processing steps, the entire synthesis can be significantly faster, meaning more coupling cycles can be achieved in a shorter period. Such advantages can have a significant, positive effect on the overhead allocation of the peptide synthesis cost, especially for longer peptides where more coupling cycles are required.
  • the present disclosure can include the utilization of amino acids and/or peptides in combination with protecting groups to form anchor molecules for solution-phase peptide synthesis.
  • amino acids can be protected at their side chains and/or termini with protecting groups (e.g. GAP protecting groups), and further coupled to one another (and/or to other molecules) to form anchor molecules.
  • protecting groups e.g. GAP protecting groups
  • the present disclosure can include a chemical composition that can include one or more group-assisted purification (GAP) constituents (in one embodiment, each GAP constituent can preferably be less than 300 Daltons); one or more amino acid (AA) constituents; one or more linker constituents; and/or one or more stopper (cap) constituents.
  • GAP constituent can be an amino-acid-GAP constituent (AA(Gap) constituent).
  • a GAP constituent and linker constituent can be GAP constituents and linker constituents (respectively) as described in U.S. App. Ser. No. 17/104,166.
  • a stopper constituent can be similar to or the same as the spacer constituents discussed in U.S. App. Ser. No. 17/104,166.
  • the present disclosure can include structures of the type H-AA(Gap)-OH, H-[AA1] ⁇ -[AA(Gap)] ⁇ -[AA 2 ] ⁇ - Stopper, H-Linker- [AA1] ⁇ -[AA(Gap)] ⁇ -[AA- 2 ] y - Stopper, and/or H-Linker- ⁇ [AA1] ⁇ -[AA(Gap)] ⁇ - [AA2] ⁇ -Stopper ⁇ , which can collectively be referred to as H-Anchor (anchor) (GAP anchor) molecules, where H can refer to the element hydrogen; O can refer to the element oxygen; a, b, and g can be any integer from 0 up to and including 10; d can be any integer from 0 up to and
  • R 4 and R 5 can both be any of H, methyl, ethyl, propyl, isopropyl, benzyl, isobutyl, sec-butyl, or tert-butyl — they can be the same or different.
  • “ can depict an attachment point of a given molecule to any other molecule.
  • a GAP constituent can include:
  • HNtBuODpp wherein can depict attachment of this GAP constituent to another molecule or atom, such as to an amino acid (e.g. with respect to an AA(Gap) constituent).
  • HNtBuODpp (or another GAP constituent) can be coupled to an amino acid side- chain or C-terminus to form an AA(Gap) constituent.
  • attaching a GAP constituent to an amino acid side chain or C-terminus can leave the C-terminus or side chain (respectively) of the amino acid free to participate in coupling reactions, such as with additional GAP-protected amino acids.
  • one or more AA(Gap) constituents can be included in an anchor molecule, or be an anchor molecule.
  • two or more AA(Gap) constituents can be coupled together, such as to form an anchor molecule or part of an anchor molecule.
  • a peptide-based anchor molecule can be formed to enable solution-phase peptide synthesis.
  • a GAP constituent can be an AA(Gap) constituent and can preferably include an amino acid coupled to a group-assisted purification molecule.
  • an AA(Gap) constituent can include a GAP constituent and an amino acid constituent.
  • an AA(Gap) constituent e.g., can include the following structures as exemplified in FIG.
  • IB (such as with respect to anchors including H-AA(Gap)-OH, H-[AA 1 ] ⁇ - [AA(Gap)] ⁇ - [ A A 2 ] ⁇ - Stopper, H-Linker- [AA 1 ] ⁇ -[AA(Gap)] ⁇ -[AA 2 ] ⁇ -Stopper), and/or H-Linker- ⁇ [ AA 1 ] ⁇ - [ A A(Gap)] ⁇ - [ AA2] ⁇ - Stopper ⁇ ⁇ : wherein R 10 can be -C(O)-, -(CH 2 )m-, or NH; R 6 , R 7 , R 8 , and R 9 can be any of -C(O)-, S, O, NH,
  • R 4 and R 5 can be H, methyl, ethyl, propyl, isopropyl, benzyl, isobutyl, sec-butyl, or tert-butyl; and j, k, m, and p can each be separate a value that can be any integer from 0 up to and including 30.
  • R 6 , R 7 , R 8 , and R 9 can all be different, and “p” can be a different value for each of R 6 , R 7 , R 8 , and R 9 .
  • an anchor can include an amino acid constituent (AA constituent), such as in addition to a GAP constituent or AA(Gap) constituent, such as with respect to anchors including 2 ' .
  • AA constituent an amino acid constituent
  • an amino acid constituent can be any molecule having a primary or secondary amine, and a carboxylic acid.
  • an amino acid constituent e.g.
  • Z 1 can be S, O, NH, NMe, Net, NBn, NPh, -C(O)-, or -(CH 2 )d-
  • Z 2 can be S, O NH, NMe, Net, NBn, NPh, -C(O)-, or -(CH 2 ) e -
  • d, e, f, g, h can each be a value that can be any integer from 0 up to and including 30
  • R 3 can be H, methyl, ethyl, propyl, isopropyl, benzyl, isobutyl, sec- butyl, tert-butyl, 2-(methylmercapto)ethyl, or:
  • an anchor can include a linker constituent (Linker) such as in addition to a GAP constituent and/or AA(Gap) constituent, such as with respect to anchors including H-AA(Gap)-OH,
  • a linker constituent can include linkers known in the in the art with respect to peptide synthesis.
  • a linker constituent e.g. ⁇ , . can include:
  • W can be S, NH, NMe, NEt, NBn, NPh, O, -C(0)-, -(CH 2 ) a2 -;
  • X can be S, NH,
  • R 1 can be -(CH 2 ) b -H, -CCl 3 , -CF 3 , phenyl, isopropyl, tert-butyl, chlorophenyl, dichlorophenyl, methoxyphenyl, or dimethoxyphenyl;
  • R 2 can be -(CH 2 ) c - H, -CCl3, -CF3, phenyl, isopropyl, tert-butyl, chlorophenyl, dichlorophenyl, methoxyphenyl, or dimethoxyphenyl; and a, b, c, and n can be separate values that can be any integer from 0 up to and including 30.
  • the “H” (hydrogen) in “H-Linker” (as used in, e.g., H- and/or Stopper ⁇ ) is attached to the “Y” variable of the Linker (such attachment notated above with a squiggly line).
  • an anchor can include a stopper constituent (Stopper) such as in addition to a GAP constituent and/or AA(Gap) constituent and/or linker constituent, such as with respect to anchors including 6.
  • a stopper constituent e.g., a stopper constituent (Stopper) such as in addition to a GAP constituent and/or AA(Gap) constituent and/or linker constituent, such as with respect to anchors including 6.
  • a stopper constituent e.g.
  • R 11 can be S, O, NH, NMe, Net, NBn, or NPh
  • R 12 , R 13 , and R 14 can be any of H, methyl, ethyl, propyl, isopropyl, benzyl, isobutyl, sec-butyl, tert-butyl, and -(CH 2 )q-H
  • R 6 , R 7 , R 8 , and R 9 can be any of -C(O)-, S, O, NH, NMe, Net, NBn, NPh, or -(CH2) q -
  • R 4 and R 5 can be any of H, methyl, ethyl, propyl, isopropyl, benzyl, isobutyl, sec-butyl, or tert-butyl
  • q can be any integer from 0 up to and including 30.
  • compositions such as compositions that can be useful as anchor molecules in accordance with the principles of the present disclosure.
  • an anchor molecule can include:
  • the anchor can follow the H-Linker- [AA 1 ] ⁇ -[AA(Gap)] ⁇ -[AA 2 ] ⁇ -Stopper) format, wherein a can be 0, b can be 2, and g can be 1.
  • each constituent can be defined as follows:
  • an anchor molecule can include:
  • the anchor can follow the H-Linker- [AA 1 ] ⁇ -[AA(Gap)] ⁇ -[AA 2 ] ⁇ -Stopper) format, wherein a can be 0, b can be 1, and g can be 0.
  • each constituent can be defined as follows:
  • an anchor molecule can include:
  • the anchor can follow the H- [AA 1 ] ⁇ -[AA(Gap)] ⁇ -[AA 2 ] ⁇ -Stopper) format, wherein a can be 0, b can be 3, and g can be 0.
  • each constituent can be defined as follows:
  • an anchor molecule can include:
  • the anchor can follow the H-Linker- ⁇ [AA 1 ] ⁇ -[AA(Gap)] ⁇ -[AA 2 ] ⁇ -Stopper) format, wherein a can be 0, b can be 0, g can be 0, and d can be 2.
  • each constituent can be defined as follows:
  • the present disclosure can include a method of synthesizing an anchor (in some embodiments, an anchor peptide).
  • the method can be modular system, such that an initial starting material (e.g., an AA(Gap) constituent) can be made in bulk, and then incorporated into an anchor to varying degrees such that the anchor can have different properties depending on the degree of incorporation of the starting material.
  • an AA(Gap) constituent can be a GAP amino acid (e.g., an amino acid with a protecting group, preferably a GAP protecting group, attached thereto) and can further act as a modular starting material.
  • a synthesis method can begin with:
  • this step can be considered an attachment of an AA(Gap) constituent to a stopper constituent (e.g. H2NC16H 33 ).
  • a stopper constituent e.g. H2NC16H 33 .
  • the above molecule can then be coupled to a first amino acid of a peptide, or alternatively, a linker constituent can be further incorporated before attachment to a first amino acid.
  • the present disclosure can further include the addition of one or more further AA(Gap) constituents to the above molecule.
  • AA(Gap) constituents for the synthesis of a longer peptide sequence (e.g., between 25 amino acids and 100 amino acids), it can be advantageous to include at least one other AA(Gap) constituent in the anchor, such that the solubility of the target peptide can be further controlled as the sequence lengthens.
  • the above molecule can be reacted with another AA(Gap) constituent (e.g., the same or different AA(Gap) constituent as used previously) to achieve:
  • the above molecule can then be coupled to a first amino acid of a peptide, or alternatively, a linker constituent can be further incorporated before attachment to a first amino acid.
  • the present disclosure can further include the addition of one or more further AA(Gap) constituents to the above molecule.
  • the above molecule can be coupled to another AA(Gap) constituent to yield:
  • the above molecule can then be coupled to a first amino acid of a peptide, or alternatively, a linker constituent can be further incorporated before attachment to a first amino acid.
  • a linker constituent can be further incorporated before attachment to a first amino acid.
  • the present disclosure can further include the addition of one or more further AA(Gap) constituents to the above molecule.
  • the method exemplified in FIG. 8 can be considered a modular system, in that one or more units (e.g. an AA(Gap) constituent) can be added multiple times to achieve greater and greater solubility control, as needed.
  • AA(Gap) constituent e.g. an AA(Gap) constituent
  • Such modularity is advantageous in the art because it enables the large-scale synthesis and stockpiling of starting materials that can then be converted into customized anchors as needed.
  • multiple different amino acids can be used in this method to lend different characteristics to the anchor.
  • the stopper constituent can be modified to lend desired characteristics, such as increased hydrophobicity, increased hydrophilicity, increased aromaticity, and/or any other desired quality.
  • Coupling and deprotection reactions can occur in one or more solvents, such as, e.g., dichloromethane, DMF, ethyl acetate, tetrahydrofuran, and/or 2- methyltetrahydrofuran.
  • solvents such as, e.g., dichloromethane, DMF, ethyl acetate, tetrahydrofuran, and/or 2- methyltetrahydrofuran.
  • the GAP, linker, amino acid, and stopper constituents can be considered as separate chemical entities capable of lending different useful properties to a given GAP anchor.
  • the GAP constituent can include a GAP molecule coupled to an amino acid that includes a polar bond (e.g. phosphine oxide) and aromatic constituents, which ultimately give the anchor a polar-organic quality with potential for pi-pi stacking, which can assist the anchor (and attached target molecule) in selectively precipitating from non-polar solvents; in another embodiment, this can assist in the anchor in excluding itself from aqueous solvents and non-polar solvents.
  • a polar bond e.g. phosphine oxide
  • aromatic constituents e.g. phosphine oxide
  • a linker constituent in the same anchor, can provide the anchor with the ability to be selectively removed from a desired substrate while additionally contributing desired solubility properties to the anchor.
  • a linker constituent can be specifically designed to form a non-labile bond at one location, and additionally form a labile bond at another location. This orthogonality can ultimately be translated to the anchor; the GAP constituent and linker constituent together can, in this manner, provide a GAP anchor with polar-organic qualities lending itself to group-assisted purification chemistry, that additionally can be cleaved under conditions the linker constituent itself would otherwise be amenable to.
  • a stopper constituent in the same (or different) anchor, can provide further desired characteristics to the anchor.
  • a stopper can be highly organic or non-polar, increasing the overall solubility of the anchor in non-polar solvents; in another example, the stopper can be highly polar, such that the anchor can also be more polar and therefore less soluble in non-polar, organic solvents.
  • an anchor can thereby be specially formulated to assist in the synthesis of a given molecule with particular characteristics.
  • a desired peptide can be synthesized with assistance from an anchor.
  • a GAP constituent (including an AA(Gap) constituent) of the anchor can have qualities discussed herein that help render the anchor (and attached peptide) soluble in organic solvents; the GAP constituent can also be amenable to pi-pi stacking to assist the peptide in selective precipitation, such as via drop precipitation. These characteristics can be further accentuated in an anchor by including more than one AA(Gap) constituent in one anchor molecule.
  • two AA(Gap) constituents can be coupled together via the termini of the amino acids therein, such as via well-known coupling reactions, to form a protected peptide.
  • this GAP-protected peptide can be incorporated into an anchor molecule for peptide synthesis.
  • a stopper constituent can also be included in the anchor — in one embodiment, a stopper constituent can include an aliphatic amine and/or alcohol and/or carboxylic acid capable of coupling to a terminus of an amino acid.
  • a linker constituent can also be included, and lend a free moiety capable of participating in a reaction, such as a coupling reaction.
  • the bond formed by the free moiety of the linker constituent can be labile to, for example, a TFA-deprotection known in the art like those discussed herein.
  • the constituents addressed herein are described separately to better communicate the different characteristics that each constituent can contribute to a given anchor molecule, and the separate descriptions of the constituents herein should not be considered as limiting.
  • any of the constituents discussed herein can be considered protecting groups.
  • GAP constituents, AA(Gap) constituents, linker constituents, and stopper constituents can attach to a given molecule and protect that area of the molecule from unwanted side reactions.
  • the anchors discussed herein can further be considered protecting groups in-and-of themselves.
  • anchors discussed herein can be attached to a C-terminus of an amino acid, such as via a nucleophilic moiety (e.g., NH, OH, etc.) that can attach a carbonyl carbon.

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Abstract

La divulgation concerne des compositions qui peuvent servir d'ancrages pour la synthèse chimique de peptides. Les molécules d'ancrage peuvent comprendre des constituants GAP, des constituants lieurs, des constituants acides aminés et/ou des constituants bloquants. Les molécules d'ancrage peuvent également comprendre des peptides d'ancrage, un peptide d'ancrage pouvant être accouplé amovible à un acide aminé d'une séquence donnée et servant d'ancrage GAP en réalisant une régulation de solubilité sur le peptide cible au fur et à mesure qu'il est synthétisé par addition d'un ou plusieurs autres acides aminés ; le peptide d'ancrage peut ensuite être retiré du peptide cible. La divulgation concerne également un nouveau procédé de synthèse de peptides qui utilise de nouvelles molécules d'ancrage et/ou des peptides d'ancrage.
EP22792712.6A 2021-04-23 2022-04-22 Compositions pour la synthèse chimique de peptides Pending EP4326735A1 (fr)

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